P600: HIGH-DEPTH RNA-SEQUENCING IDENTIFIES CD8+ STAT3 MUTATED T-LGLL PATIENTS AS A DISTINCT BIOLOGICAL ENTITY WITHIN THE DISEASE HETEROGENEITY
Authors: G. Calabretto, A. Binatti, A. Teramo, G. Barilà, A. Buratin, V. R. Gasparini, C. Vicenzetto, E. Gaffo, V. Trimarco, L. Trentin, M. Facco, F. Vianello, G. Semenzato, R. Zambello, S. Bortoluzzi
Journal: HemaSphere (2022)

Background: T-Large granular lymphocyte leukemia (T-LGLL) is a rare lymphoproliferative disorder characterized by the clonal expansion of cytotoxic T-LGL. We recognized a most common CD8+ subtype (CD8+ T-LGLL) and a CD4+ variant (CD4+ T-LGLL). STAT3 and STAT5B activating mutations, the main genetic lesions, are linked to the immunophenotype of the leukemic clone, T-LGLL clinical manifestations and disease prognosis. To schematize the biological heterogeneity of the disease, 4 main T-LGLL subsets can be identified: CD8+ STAT3M, CD8+ WT, CD4+ STAT5BM and CD4+ WT (M, mutated; WT, wild-type). Although STAT3 and STAT5b may regulate the transcription of genes involved in disease severity, the relationship between T-LGL immunophenotype (CD8+ or CD4+), STATs mutational status and gene expression profiles (GEP), including non-coding-RNAs, has never been investigated. Aims: The aim of the study was to disclose T-LGLL transcriptome unbalances accounting for disease pathogenesis and clinical manifestations, by investigating GEP of leukemic T-LGL from each disease subtype and healthy controls (CTR). Methods: High-depth RNA-seq was performed with a HiSeq3000 (Illumina) on RNA extracted from immuno-magnetically purified T-LGL of 20 T-LGLL patients (stratified in the 4 groups) and 5 CTR; 150 paired-end reads were generated (315 million reads/sample, on average). RNA-seq data were analyzed for reads alignment and transcripts quantification by CircComPara pipeline. Differentially expressed genes (DEGs) were assessed using DESeq (adj. p<0.01) and validated by RT-qPCR. Results: Unsupervised multi-dimensional scaling analysis of GEP clearly separated T-LGLL from CTR samples. Of note, CD8+ STAT3M patients, characterized by a symptomatic disease and reduced survival, resulted as a distinct biological entity with respect to the other T-LGLL subgroups, that showed similar transcriptomic features. In detail, we found 1612 DEGs in CD8+ STAT3M and 1707 DEGs in the other T-LGLL cases, with the majority being down-regulated as compared to CTR. Of these, 641 genes were commonly dysregulated in all T-LGLL subgroups, although a more marked dysregulation was observed in presence of STAT3 mutations. 971 genes were differentially expressed uniquely in CD8+ STAT3M cases, with 571 being differentially expressed also versus other T-LGLL subgroups. Overall, dysregulated genes included tumor suppressors, protein kinases and phosphatases, cytokines and receptors. Of note, two non coding-RNAs with a known oncogenic role in other malignancies were up-regulated in LGLL samples. Next, Gene Set Enrichment Analyses (GSEA) disclosed pathway dysregulation. In CD8+ STAT3M cases proteasome and Interferon signaling emerged among the most aberrantly activated signaling pathways with respect to CTR, whereas the cell cycle checkpoints pathway resulted to be activated as compared to the other T-LGLL subgroups. Considering CD4+ STAT5BM T-LGLL, we observed the upregulation of a JAK/STAT axis negative regulator, reported to be down-expressed in other STAT5B-related disorders characterized by poor prognosis. This transcriptional signature might account for the peculiar indolent clinical course of STAT5BM T-LGLL patients. Summary/Conclusion: We identified new genetic players involved in T-LGLL pathogenesis and disease severity, showing a peculiar GEP of CD8+ STAT3M cases, which are the most symptomatic and treatment-requiring patients. Most importantly, the identified DEGs are disclosing novel potential therapeutic targets to design innovative RNA-based therapies for T-LGLL patients.